Compositions and methods for modification of biomolecules

Patent metadata

Field	Value
Publication number	US-9260371-B2
Application number	US-201313868444-A
Country	US
Kind code	B2
Filing date	Apr 23, 2013
Priority date	Nov 1, 2004
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

The present invention provides modified cycloalkyne compounds; and method of use of such compounds in modifying biomolecules. The present invention features a cycloaddition reaction that can be carried out under physiological conditions. In general, the invention involves reacting a modified cycloalkyne with an azide moiety on a target biomolecule, generating a covalently modified biomolecule. The selectivity of the reaction and its compatibility with aqueous environments provide for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

First claim

What is claimed is:

1. A compound of the formula:

wherein each of R 1 , R 2 , R 5 and R 6 is independently H or a halogen atom; and
R 3 is linked to R 4 through a C, O, N or S atom to form a substituted cycloalkyl or heterocycloalkyl ring which optionally is linked to a molecule of interest or a moiety that comprises a reactive group that facilitates covalent attachment of a molecule of interest.

2. The compound of claim 1 , wherein R 3 is linked to R 4 through a carbon atom thus forming a substituted cycloalkyl ring.

3. The compound of claim 2 , wherein the substituted cycloalkyl ring is a substituted cyclopropyl ring.

4. The compound of claim 2 , wherein the substituted cycloalkyl ring is linked to a reactive group that facilitates covalent attachment of a molecule of interest, wherein the reactive group is selected from the group consisting of a carboxyl, an amine, an ester, a thioester, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide and a hydrazine.

5. The compound of claim 2 , wherein the substituted cycloalkyl ring is linked to a molecule of interest selected from the group consisting of a detectable label, a toxin, a linker, a peptide, a drug, a member of a specific binding pair and an epitope tag.

6. The compound of claim 1 wherein R 1 , R 2 , R 5 and R 6 are each H; and R 3 is linked to R 4 through a carbon atom thus forming a substituted cycloalkyl ring.

7. The compound of claim 6 , wherein the substituted cycloalkyl ring is a substituted cyclopropyl ring.

8. The compound of claim 6 , wherein the substituted cycloalkyl ring is linked to a moiety that comprises a reactive group selected from the group consisting of a carboxyl, an amine, an ester, a thioester, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide and a hydrazine.

9. The compound of claim 6 , wherein the substituted cycloalkyl ring is linked to a moiety that comprises a molecule of interest selected from the group consisting of a detectable label, a toxin, a linker, a peptide, a drug, a member of a specific binding pair and an epitope tag.

10. A method for chemoselective modification of a target molecule comprising an azide, the method comprising reacting an azide of a target molecule with the compound of claim 1 , wherein said reacting produces a conjugate between the azide of the target molecule and the compound.

11. The method of claim 10 , wherein the target molecule is a sugar.

12. The method of claim 11 , wherein the sugar is a substrate of sialic acid biosynthesis.

13. The method of claim 11 , wherein the sugar is mannosamine or acetylated mannosamine.

14. The method of claim 10 , wherein the target molecule is an amino acid.

15. The method of claim 10 , wherein the target molecule comprising the azide is expressed on a cell surface.

16. A method for synthetically modifying a cellular component, the method comprising:
introducing an azide moiety into a cellular component, thereby generating an azide-modified cellular component; and
contacting the cell comprising the azide-modified cellular component with a reactive partner comprising the compound of claim 1 , said contacting being under physiological conditions;
wherein said contacting with said reactive partner results in reaction between the azide group of azide-modified cellular component and the compound of claim 1 , thereby synthetically and covalently modifying the cellular component.

Assignees

• Univ California

Inventors

• Bertozzi Carolyn Ruth
• Agard Nicholas J
• Prescher Jennifer A
• Baskin Jeremy Michael
• Sletten Ellen May

Classifications

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• C07C63/74

  having unsaturation outside the aromatic rings · CPC title

• C07C45/63

  by introduction of halogen; by substitution of halogen atoms by other halogen atoms · CPC title

• C07D495/04

  Ortho-condensed systems · CPC title

• C07C2601/18

  with a ring being at least seven-membered · CPC title